Targeted cancer therapies that can selectively kill cancer cells without harming other cells in the body would represent a major improvement in the clinical treatment of cancer. It would be highly desirable to develop a strategy to directly target cancer cells with chemotherapeutic agents in cancer treatment regimens. This could lead to reduction or elimination of toxic side effects, more efficient delivery of the drug to the targeted site, and reduction in dosage of the administered drug and a resulting decrease in toxicity to healthy cells and in the cost of the chemotherapeutic regimen. Reports of targeting chemotherapeutic drugs using antibodies have appeared in the literature since 1958. Targeting drugs by conjugation to antibodies for selective delivery to cancer cells has had limited success due to the large size of antibodies (MW=125-150 kilodaltons or KD) and thus their relative inability to penetrate solid tumors. An alternative strategy comprises the use of smaller targeting ligands and peptides, which recognize specific receptors unique to or overexpressed on tumor cells, as the targeting vector. Such constructs have molecular weights of 2-6 KD, which allow ready penetration throughout solid tumors.
Increased cell proliferation and growth is a trademark of cancer. The increase in cellular proliferation is associated with high turnover of cell cholesterol. Cells requiring cholesterol for membrane synthesis and growth may acquire cholesterol by receptor mediated endocytosis of plasma low density lipoproteins (LDL), the major transporter of cholesterol in the blood, or by de novo synthesis. LDL is taken up into cells by a receptor known as the LDL receptor (LDLR); the LDL along with the receptor is endocytosed and transported into the cells in endosomes. The endosomes become acidified and this releases the LDL receptor from the LDL; the LDL receptor recycles to the surface where it can participate in additional uptake of LDL particles. There is a body of evidence that suggests that tumors in a variety of tissues have a high requirement for LDL to the extent that plasma LDLs are depleted. The increased import of LDL into cancerous cells is thought to be due to elevated LDL receptors (LDLR) in these tumors. Some tumors known to express high numbers of LDLRs include some forms of leukemia, lung tumors, colorectal tumors and ovarian cancer. In vivo studies showed that LDLRs do appear in brain malignancies. Leppala et al used PET imaging, and demonstrated that 99 mTc_LDL localizes in human brain tumors in vivo but not in normal brain.
This suggests that the LDL receptor is a potential unique molecular target in GBM and other malignancies for the delivery of anti-tumor drugs via LDL particles. A test of this possibility was undertaken by Maranhão and coworkers. A protein-free microemulsion (LDE) with a lipid composition resembling that of low-density lipoprotein (LDL) was used in metabolic studies in rats to compare LDE with the native lipoprotein. Incubation studies also showed that LDE incorporates a variety of apolipoproteins, including apo E, a ligand for recognition of lipoproteins by specific receptors.
Lipophilic Derivatives of Cancer Chemotherapeutic Agents:
Arbor Therapeutics has developed unique lipophilic derivatives of the cancer chemotherapeutic agent which have high stability in normal systemic circulation and retention in the lipid core of the LDL particles but readily release the active chemotherapeutic agent in the acidic environment of the endosome. See U.S. Pat. No. 8,440,714, the disclosure of which is incorporated herein in its entirety.
In another embodiment, there is provided a active chemotherapeutic compounds of the formula 3a or 3b:
wherein: R1 is hydrogen, C1-C4 alkyl or C5-C22 alkyl; R2 is C5-C22 alkyl; Y is selected from O, NR′ or S wherein R′ is hydrogen or C1-C6 alkyl; Z is selected from O or S; Q is O or S; and T is O or S. In one aspect of the compound, R1 is hydrogen or C1-C4 alkyl; R2 is C5-C22 alkyl; Y is O or S; Z is O; Q is O; and T is O. The activated compound of the formula 3a or 3b may be used to prepare the acid labile lipophilic conjugate when the activated compound is condensed with a hydroxyl bearing cancer chemotherapeutic agent (HBCCA). As defined herein, the HBCCA is represented generically with the residue or group “R” in the formulae 1, 1a, 1b, 1.1, 2 and 2a, for example, and where the HBCCA is not coupled to form the acid labile, lipophilic molecular conjugates, then the HBCCA may also be generically represented as having the formula “R—OH” since the HBCCA may be functionalized by one or more hydroxyl (—OH) groups.
In one embodiment, there is provided an acid labile lipophilic molecular conjugate (ALLMC) of the formula 1, 1.1 or formula 2:
wherein: R is a hydroxyl bearing cancer chemotherapeutic agent; for formula 1 or 1.1 R1 is hydrogen, C1-C4 alkyl or C5-C22 alkyl; R2 is C5-C22 alkyl; Y is selected from O, NR′ or S wherein R′ is hydrogen or C1-C6 alkyl; Z is O or S; Q is O or S; and T is O or S; for formula 2: R2 is a C1-C22 alkyl; T is O or S; and X is hydrogen or a leaving group selected from the group consisting of mesylates, sulfonates and halogen (Cl, Br and I); and their isolated enantiomers, diastereoisomers or mixtures thereof, or a pharmaceutically acceptable salt thereof. The compound 1.1 includes the pure syn isomer, the pure anti isomer and mixtures of syn- and anti-isomers, and their diastereomers.
In another embodiment, there is provided the above acid labile lipophilic molecular conjugate of the formula 1 or 1.1 wherein: R is a hydroxyl bearing cancer chemotherapeutic agent; R1 is hydrogen, C1-C4 alkyl or C5-C22 alkyl; R2 is C5-C22 alkyl; Y is O or S; Z is O; Q is O; and T is O. In one aspect of the acid labile lipophilic molecular conjugate of the formula 2 wherein: R2 is C5-C22 alkyl; T is O; and X is hydrogen or selected from the group consisting of Cl, Br and I. In another variation, R2 is C9-C22. In another aspect of the above acid labile lipophilic molecular conjugate comprising the formula 1a, 1b or formula 2a:
wherein: R is a hydroxyl bearing cancer chemotherapeutic agent (HBCCA); for formula 1a or 1b R1 is hydrogen, C1-C4 alkyl or C5-C22 alkyl; and R2 is C5-C22 alkyl; and for formula 2a: R2 is C1-C22 alkyl; and X is hydrogen or is selected from the group consisting of Cl, Br and I. In one variation of the compound that is the carbonate (i.e., —OC(O)O—) of the formula 1a or 1b the compound is the corresponding sulfonate (i.e., —OS(O)O—) of the formula 1a wherein the carbonate group is replaced by a sulfonate group. The compound 1b includes the pure syn isomer, the pure anti isomer and mixtures of syn and anti isomers, and their diastereomers.
In another variation of the compound of the formula 1, 2, 1a and 2a, R1 is hydrogen or C1-C4 alkyl or C5-C22 alkyl, and R2 is the carbon residue of an unsaturated fatty acid, such as the carbon residue selected from the group consisting of the C19 residue of eicosenoic acid (including the cis isomer, trans isomer and mixtures of isomers), C17 residue of oleic acid and the C17 residue of elaidic acid. As used herein, the “carbon residue” (e.g., C17 residue, C19 residue etc . . . ) of the fatty acid means the carbon chain of the fatty acids excluding the carboxyl carbon.
In another aspect of the above acid labile lipophilic molecular conjugate, the hydroxyl bearing cancer chemotherapeutic agent is selected from the group consisting of taxanes, abeo-taxanes, camptothecins, epothilones, cucurbitacins, quassinoids, anthracyclines, and their analogs and derivatives. In another aspect of the above acid labile lipophilic molecular conjugate, the hydroxyl bearing cancer chemotherapeutic agent is selected from the group consisting of aclarubicin, camptothecin, masoprocol, paclitaxel, pentostatin, amrubicin, cladribine, cytarabine, docetaxel, gemcitabine, elliptinium acetate, epirubicin, etoposide, formestane, fulvestrant, idarubicin, pirarubicin, topotecan, valrubicin and vinblastine.
In one embodiment, there is provided an acid labile lipophilic molecular conjugate (ALLMC) of the formula 1, 1.1 or formula 2:
wherein: R is a hydroxyl bearing cancer chemotherapeutic agent; for formula 1 or 1.1 R1 is hydrogen, C1-C4 alkyl or C5-C22 alkyl; R2 is C5-C22 alkyl; Y is selected from O, NR′ or S wherein R′ is hydrogen or C1-C6 alkyl; Z is O or S; Q is O or S; and T is O or S; for formula 2: R2 is a C1-C22 alkyl; T is O or S; and X is hydrogen or a leaving group selected from the group consisting of mesylates, sulfonates and halogen (Cl, Br and I); and their isolated enantiomers, diastereoisomers or mixtures thereof, or a pharmaceutically acceptable salt thereof. The compound 1.1 includes the pure syn isomer, the pure anti isomer and mixtures of syn- and anti-isomers, and their diastereomers.
In another embodiment, there is provided the above acid labile lipophilic molecular conjugate of the formula 1 or 1.1 wherein: R is a hydroxyl bearing cancer chemotherapeutic agent; R1 is hydrogen, C1-C4 alkyl or C5-C22 alkyl; R2 is C5-C22 alkyl; Y is O or S; Z is O; Q is O; and T is O. In one aspect of the acid labile lipophilic molecular conjugate of the formula 2 wherein: R2 is C5-C22 alkyl; T is O; and X is hydrogen or selected from the group consisting of Cl, Br and I. In another variation, R2 is C9-C22. In another aspect of the above acid labile lipophilic molecular conjugate comprising the formula 1a, 1b or formula 2a:
wherein: R is a hydroxyl bearing cancer chemotherapeutic agent (HBCCA);for formula 1a or 1b R1 is hydrogen, C1-C4 alkyl or C5-C22 alkyl; and R2 is C5-C22 alkyl;    and for formula 2a: R2 is C1-C22 alkyl; and X is hydrogen or is selected from the group consisting of Cl, Br and I. In one variation of the compound that is the carbonate (i.e., —OC(O)O—) of the formula 1a or 1b the compound is the corresponding sulfonate (i.e., —OS(O)O—) of the formula 1a wherein the carbonate group is replaced by a sulfonate group. The compound 1b includes the pure syn isomer, the pure anti isomer and mixtures of syn and anti isomers, and their diastereomers.
In another variation of the compound of the formula 1, 2, 1a and 2a, R1 is hydrogen or C1-C4 alkyl or C5-C22 alkyl, and R2 is the carbon residue of an unsaturated fatty acid, such as the carbon residue selected from the group consisting of the C19 residue of eicosenoic acid (including the cis isomer, trans isomer and mixtures of isomers), C17 residue of oleic acid and the C17 residue of elaidic acid. As used herein, the “carbon residue” (e.g., C17 residue, C19 residue etc . . . ) of the fatty acid means the carbon chain of the fatty acids excluding the carboxyl carbon. In another aspect of the above acid labile lipophilic molecular conjugate, the hydroxyl bearing cancer chemotherapeutic agent is selected from the group consisting of taxanes, abeo-taxanes, camptothecins, epothilones, cucurbitacins, quassinoids, anthracyclines, and their analogs and derivatives. In another aspect of the above acid labile lipophilic molecular conjugate, the hydroxyl bearing cancer chemotherapeutic agent is selected from the group consisting of aclarubicin, camptothecin, masoprocol, paclitaxel, pentostatin, amrubicin, cladribine, cytarabine, docetaxel, gemcitabine, elliptinium acetate, epirubicin, etoposide, formestane, fulvestrant, idarubicin, pirarubicin, topotecan, valrubicin and vinblastine. In another aspect of the above acid labile lipophilic molecular conjugate, the conjugate is selected from the compounds in FIGS. 18, 19 and 20. In one variation, only one of the groups -ALL1, -ALL2, -ALL3 . . . to -ALLn is an -ALL group and the others are hydrogens. In another variation, two of the groups -ALL1, -ALL2, -ALL3 . . . to -ALLn are -ALL groups.
In another embodiment, there is provided a pharmaceutical composition comprising: a) a therapeutically effective amount of a compound of the above, in the form of a single diastereoisomer; and b) a pharmaceutically acceptable excipient. In another aspect, the pharmaceutical composition is adapted for oral administration; or as a liquid formulation adapted for parenteral administration. In another aspect, the composition is adapted for administration by a route selected from the group consisting of orally, parenterally, intraperitoneally, intravenously, intraarteriall, transdermally, intramuscularly, rectally, intranasally, liposomally, subcutaneously and intrathecally. In another embodiment, there is provided a method for the treatment of cancer in a patient comprising administering to the patient a therapeutically effective amount of a compound or composition of any of the above compound or composition, to a patient in need of such treatment. In one aspect of the method, the cancer is selected from the group consisting of leukemia, neuroblastoma, glioblastoma, cervical, colorectal, pancreatic, renal and melanoma. In another aspect of the method, the cancer is selected from the group consisting of lung, breast, prostate, ovarian and head and neck. In another aspect of the method, the method provides at least a 10%, 20%, 30%, 40%, or at least a 50% diminished degree of resistance expressed by the cancer cells when compared with the non-conjugated hydroxyl bearing cancer chemotherapeutic agent.
In another embodiment, there is provided a method for reducing or substantially eliminating the side effects of chemotherapy associated with the administration of a cancer chemotherapeutic agent to a patient, the method comprising administering to the patient a therapeutically effective amount of an acid labile lipophilic molecular conjugate of the formula 1, 1.1 or formula 2:

wherein: R is a hydroxyl bearing cancer chemotherapeutic agent; for formula 1 or 1.1: R1 is hydrogen, C1-C4 alkyl or C5-C22 alkyl; R2 is C5-C22 alkyl; Y is selected from O, NR′ or S wherein R′ is hydrogen or C1-C6 alkyl; Z is O or S; Q is O or S; and T is O or S; for formula 2: R2 is C1-C22 alkyl; T is O or S; and X is hydrogen or a leaving group selected from the group consisting of mesylates, sulfonates and halogen (Cl, Br and I); and their isolated enantiomers, diastereoisomers or mixtures thereof. The compound 1.1 includes the pure syn isomer, the pure anti isomer and mixtures of syn and anti isomers, and their diastereomers. In one variation of the above, R2 is C9-C22 alkyl. In one aspect, the method provides a higher concentration of the cancer chemotherapeutic agent in a cancer cell of the patient. In another aspect, the method delivers a higher concentration of the cancer chemotherapeutic agent in the cancer cell, when compared to the administration of a non-conjugated cancer chemotherapeutic agent to the patient, by at least 5%, 10%, 20%, 30%, 40% or at least 50%.
In another embodiment, there is provided a compound of the formula 3a or 3b:

wherein: R1 is hydrogen, C1-C4 alkyl or C5-C22 alkyl; R2 is C5-C22 alkyl; Y is selected from O, NR′ or S wherein R′ is hydrogen or C1-C6 alkyl; Z is selected from O or S; Q is O or S; and T is O or S. In one aspect of the compound, R1 is hydrogen or C1-C4 alkyl; R2 is C5-C22 alkyl; Y is O or S; Z is O; Q is O; and T is O. The activated compound of the formula 3a or 3b may be used to prepare the acid labile lipophilic conjugate when the activated compound is condensed with a hydroxyl bearing cancer chemotherapeutic agent (HBCCA). As defined herein, the HBCCA is represented generically with the residue or group “R” in the formulae 1, 1a, 1b, 1.1, 2 and 2a, for example, and where the HBCCA is not coupled to form the acid labile, lipophilic molecular conjugates, then the HBCCA may also be represented as having the formula “R—OH” since the HBCCA may be functionalized by one or more hydroxyl (—OH) groups.
Similarly, the acid labile lipophilic group (i.e., the “-ALL” group of the activated compound) that may be condensed with a HBCCA to form the acid labile, lipophilic molecular conjugate generically represented as “R-O-ALL.” Accordingly, where more than one -ALL group is condensed or conjugated with a HBCCA group, then each -ALL group may be independently designated as -ALL1, -ALL2, -ALL3 . . . to -ALLn where n is the number of available hydroxyl groups on the cancer chemotherapeutic agent that may be conjugated or couple with an -ALL group. As exemplified for the compound of formulae 1 and 2, for example, the HBCCA and the -ALL groups as designated, are shown below.

An example of an acid labile, lipophilic molecular conjugate (ALLMC), where the HBCCA group is paclitaxel having two -ALL groups, is depicted below:

In the above representative example of the acid labile molecular conjugate of paclitaxel, each of the -ALL1 and -ALL2 is independently hydrogen or an -ALL group as defined herein. For HBCCA groups having more than one hydroxyl groups, the inaccessible hydroxyl group or groups where the acid labile lipophilic group cannot be formed, then the group that is designated as an -ALL group(s) is hydrogen.
In another aspect of the above acid labile lipophilic molecular conjugate, the hydroxyl bearing cancer chemotherapeutic agent is selected from the group consisting of taxanes, abeo-taxanes, camptothecins, epothilones, cucurbitacins, quassinoids, anthracyclines, and their analogs and derivatives. In another aspect of the above acid labile lipophilic molecular conjugate, the hydroxyl bearing cancer chemotherapeutic agent is selected from the group consisting of aclarubicin, camptothecin, masoprocol, paclitaxel, pentostatin, amrubicin, cladribine, cytarabine, docetaxel, gemcitabine, elliptinium acetate, epirubicin, etoposide, formestane, fulvestrant, idarubicin, pirarubicin, topotecan, valrubicin and vinblastine.
Representative chemotherapeutic agents that may be employed in the present composition or formulations are disclosed in Figures A, B and C. In one aspect of the above, the chemotherapeutic agent is ART-207.

Capturing the great potential of selective and specific delivery of chemotherapeutic compounds to cancer tissues via their over expression of LDL receptors and consequent high uptake of LDL particles from the systemic circulation, requires that the cancer chemotherapeutic agent have high lipophilicity so as to remain entrapped in the lipid core of the LDL particle and not diffuse into the plasma to lead to toxic side effects from exposure of normal tissues to the agent. Further, once the LDL particle with its chemotherapeutic payload has entered the cancer cell via LDL receptor mediated uptake into the acidic environment of the endosome, the LDL receptor is disassociated from the LDL particle and is recycled to the cell surface and the LDL particle releases its lipid contents and its lipophilic chemotherapeutic agent to the enzymes and acidic environment of the endosome.
Further validity of this expectation was shown by Maranhão and coworkers who demonstrated that a cholesterol-rich microemulsion or nanoparticle preparation (LDE) concentrates in cancer tissues after injection into the bloodstream. The cytotoxicity, pharmacokinetics, toxicity to animals and therapeutic action of a paclitaxel lipophilic derivative associated to LDE were compared with those of commercial paclitaxel. Results showed that LDE-paclitaxel oleate was stable. The cytostatic activity of the drug in the complex was diminished compared with the commercial paclitaxel due to the cytotoxicity of the vehicle Cremophor EL used in the commercial formulation. Competition experiments in neoplastic cultured cells showed that paclitaxel oleate and LDE are internalized together by the LDL receptor pathway. Tolerability to mice was remarkable, such that the lethal dose (LD50) was nine fold greater than that of the commercial formulation (LD50=326 μM and 37 μM, respectively). LDE concentrates paclitaxel oleate in the tumor roughly fourfold relative to the normal adjacent tissues. At equimolar doses, the association of paclitaxel oleate with LDE resulted in remarkable changes in the drug pharmacokinetic parameters when compared to commercial paclitaxel (t1/2=218 min and 184 min, AUC=1,334 μg-h/mL and 707 μg-h/mL and CL=0.125 mL/min and 0.236 mL/min, respectively). The therapeutic efficacy of the complex was pronouncedly greater than that of the commercial paclitaxel, as indicated by the reduction in tumor growth, increase in survival rates and % cure of treated mice. Maranhão et al showed LDE-paclitaxel oleate is a stable complex and compared with paclitaxel, toxicity is considerably reduced and activity is enhanced which may lead to improved therapeutic index in clinical use. Maranhão and coworkers followed up their preliminary animal studies with a pilot clinical study in breast cancer patients. The clinical study was performed in breast cancer patients to evaluate the tumoral uptake, pharmacokinetics and toxicity of paclitaxel associated to LDE nanoemulsions. Twenty-four hours before mastectomy 3H-paclitaxel oleate associated with 14C-cholesteryl oleate-nanoemulsion or 3H-paclitaxel in Cremophor EL were injected into five patients for collection of blood samples and fragments of tumor and normal breast tissue. A pilot clinical study of paclitaxel-nanoemulsion administered at 3-week intervals was performed in four breast cancer patients with refractory advanced disease at 175 and 220 mg/m2 dose levels. The half-life (t1/2) of paclitaxel oleate associated to the nanoemulsion was longer than that of paclitaxel (t1/2=15.4±4.7 and 3.5±0.80 h, respectively). Uptake of the 14C-cholesteryl ester nanoemulsion and 3H-paclitaxel oleate by breast malignant tissue was threefold greater than the normal breast tissue and toxicity was minimal at the two dose levels. Their results suggest that the paclitaxel-nanoemulsion preparation can be advantageous for use in the treatment of breast cancer because the pharmacokinetic parameters are improved, the drug is concentrated in the neoplastic tissue and the toxicity of paclitaxel is reduced. Additional reports from the Maranhão laboratory of small human trials with the LDL-like lipid emulsion show that lipophilic drugs incorporated into the core of the emulsion are targeted to tumor tissue and side effects are significantly reduced. The difficulty of preparation of the emulsion, manufacture by long term sonication and extended centrifugation for particle size selection precluded them from further clinical exploration and development.
We have discovered how to prepare a nanoparticulate “pseudo LDL” lipid microemulsion as a delivery formulation for sufficiently lipophilic chemotherapeutics, including our unique acid labile, lipophilic prodrug derivative of the cancer chemotherapeutic agent. In one embodiment, the lipophilic chemotherapeutic agents have a measured or calculated Log P of greater than 4. We further demonstrate in animal tumor models that the acid labile, lipophilic molecular conjugates of cancer chemotherapeutic agent when dosed in a nanoparticulate, LDL-like lipid emulsion, is more useful for tumor reduction due to reduced toxicity and greater efficacy due to selective delivery to neoplastic/tumor tissue.
In one embodiment, the application discloses a stable, synthetic low density lipoprotein (LDL) nanoparticle comprising: a) a lipophilic anti-cancer agent; b) phospholipids (PL); and c) triglycerides (TG); wherein the LDL nanoparticle has a particle size less than 100 nm, less than 90 nm or less than 80 nm. As referred to herein, a stable synthetic low density lipoprotein (LDL) nanoparticle is a nanoparticle as defined herein that has a shelf life at about 25° C. of greater than 90 days, greater than 120 days, greater than 180 days, or greater than 1 year when stored in a sealed container and away from exposure to light. In another aspect, the nanoparticle has a shelf life at about 25° C. that is more than 1 year, or about 2 years or more when stored in a sealed container and away from exposure to light. In one aspect if the LDL nanoparticle, the particle size distribution is between 40 to 80 nm. In another aspect, the particle size distribution is between 50 and 60 nm. In one aspect of the above, the LDL nanoparticle has a mean size distribution of 60 nm. In another aspect, the LDL nanoparticle has a mean size distribution of about 50 nm. In another aspect, the phospholipids is selected from the group consisting of phosphotidylcholine, phosphotidylethanolamine, symmetric or asymmetric 1,2-diacyl-sn-glycero-3-phosphorylcholines, 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine,1,2-dimyristoyl-sn-glycero-3-phosphorylethanolamine, egg phospholipids, egg phosphatidyl glycerol, dipalmitoylphosphatidylglycerol, egg lecithin, soy lecithin, lecithin (NOS) and mixtures thereof.
In another aspect, the LDL nanoparticle further comprises cholesterol ester (CE) or cholesterol (C), or mixtures of cholesterol ester and cholesterol. In another aspect, the cholesterol ester is selected from the group consisting of C16-22 esters of cholesterol, cholesterol and mixtures thereof; and the triglycerides is selected from the group consisting of soybean oil, triolein, glyceryl tripalmitate and mixtures thereof. In one aspect of the above, the esters of cholesterol is selected from the group consisting of cholesteryl oleate, cholesteryl palmatate, cholesteryl stearate and cholesteryl lenolenate. In another aspect, the LDL nanoparticle further comprises an agent selected from the group consisting of triolein, natural antioxidants, BHT, ubiquinol, ubiquinol 10, vitamin E, alpha-tocopherol, gamma-tocopherol, lycopene, retinyl derivative and betacarotene, or mixtures thereof. In another aspect, the lipophilic anti-cancer agent is an anti-cancer agent or a prodrug of the anti-cancer agent. In one aspect of the above, the ratio of PL:TG may range from 8:1 to 3:1. In another aspect, the ratio of PL:TG:CE may range from 8:1:0.5 to 3:1:0.1. In another aspect, the ratio of the lipophilic anti-cancer agent: PL:TG may range from 1:10:3 to 1:3:0.5. In another aspect, the ratio of the lipophilic anti-cancer agent:PL:TG:CE may range from 1:10:3:1 to 1:3:0.5:0.1.
In another aspect, the anti-cancer agent is selected from the group consisting of a taxane, abeo-taxane, camptothecin, epothilone, cucurbitacin, quassinoid and an anthracycline. In another aspect, the anticancer agent is selected from the group consisting of aclarubicin, camptothecin, masoprocol, paclitaxel, pentostatin, amrubicin, cladribine, cytarabine, docetaxel, gemcitabine, elliptinium acetate, epirubicin, etoposide, formestane, fulvestrant, idarubicin, pirarubicin, topotecan, valrubicin and vinblastine. In yet another aspect, the pro-drug of the anti-cancer agent is an acid labile lipophilic molecular conjugates is as disclosed herein, and in Figures A, B and C. In one particular aspect, the acid labile lipophilic molecular conjugates is ART-207.

In another aspect of the above, the lipophilic anti-cancer agent has a log P greater than 4.0, 6.0 or 8.0. In one aspect, the weight ratio of PL:TG:CE:C ranges from 73:12:2:1 to 78:12:2:1; optionally further comprising an additive selected from the group consisting of triolein, natural antioxidants, BHT, ubiquinol, ubiquinol 10, vitamin E, alpha-tocopherol, gamma-tocopherol, lycopene, retinyl derivative and betacarotene, or mixtures thereof. In one variation, the weight ratio of PL:TG:CE:C is 77:10:2:1. In one aspect, the natural antioxidant is selected from Coenzyme Q10, resveratrol, pterostilbene and mixtures thereof. In another aspect, the ratio of the lipophilic anti-cancer agent to the triglyceride is from 1:1 to 0.6:1. In another aspect, the LDL nanoparticle contains a total solids content of 6.0 to 8% wt/wt. In another aspect, the LDL nanoparticle contains a total lipid content of 5.0 to 7.0% wt/wt. In one variation, the LDL nanoparticle further comprises a poloxamer selected from the group consisting of P188, P237, P338, P407, SYNPERONICS, PLURONICS and KOLLIPHOR, or mixtures thereof.
In another embodiment, there is provided a process for preparing a stable, synthetic low density lipoprotein (LDL) nanoparticle comprising: a) a lipophilic anti-cancer agent; b) phospholipids (PL); and c) triglycerides (TG); the process comprising: 1) combining the lipophilic anti-cancer agent, phospholipids and triglycerides to form a mixture; 2) homogenizing the mixture by dissolution in a volatile solvent; 3) removing the solvent; 4) forming a coarse emulsion by blending of the mixture in a buffer to form an emulsion mixture; 5) microfluidizing the emulsion mixture in a microfluidizer apparatus for a sufficient amount of time to produce a particle preparation of 100 nm or less; and 6) sterilizing the nanoparticle preparation through a 0.22 micron filter to obtain the synthetic LDL nanoparticles with a range of 40 nm to 80 nm. In onve variation, the synthetic low density lipoprotein (LDL) nanoparticle mixture wherein the phospholipids is selected from the group consisting of phosphotidylcholine, phosphotidylethanolamine, symmetric or asymmetric 1,2-diacyl-sn-glycero-3-phosphorylcholines, 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine,1,2-dimyristoyl-sn-glycero-3-phosphorylethanolamine, egg phospholipids, egg phosphatidyl glycerol, dipalmitoylphosphatidylglycerol, egg lecithin, soy lecithin, lecithin (NOS) and mixtures thereof. In another aspect, the LDL nanoparticle further comprises cholesterol (C) or cholesterol ester (CE) selected from the group consisting of C16-22 esters of cholesterol, cholesterol and mixtures thereof; and the triglycerides is selected from the group consisting of soybean oil, triolein, glyceryl tripalmitate and mixtures thereof; or mixtures of cholesterol and cholesterol esters. In one aspect of the above, the slow speed blending is performed at a speed of between 200 and 800 rpm, about 200 rpm, 400 rpm, 600 rpm or 800 rpm. In another aspect, the microfluidizing of the warm coarse emulsion mixture is performed at a processing temperature of about 45 to 65° C. In another aspect of the above process, the solvent is removed in vacuum. In another embodiment, there is provided a stable, synthetic low density lipoprotein (LDL) nanoparticle comprising: a) a lipophilic anti-cancer agent; b) phospholipids (PL); and c) triglycerides (TG) prepared by the process as disclosed herein. In one embodiment of the above, the synthetic LDL nanoparticle is prepared by any of the disclosed process, wherein the LDL nanoparticle becomes coated with apolipoprotein upon intra venous injection and are recognized and internalized by cellular LDL receptors.
In another embodiment, there is provided a method for the treatment of cancer in a patient comprising administering to the patient a therapeutically effective amount of the stable, synthetic low density lipoprotein (LDL) nanoparticle of any one of the above embodiments, aspects and variations, to a patient in need of such treatment. In another aspect of the method, the cancer is selected from the group consisting of leukemia, neuroblastoma, glioblastoma, cervical, colorectal, pancreatic, renal melanoma, lung, breast, prostate, ovarian and head and neck.